Single receptor assays for immunosuppressive drugs

a single receptor and immunosuppressive technology, applied in the field of immunosuppressive drug detection and quantification assays, to achieve the effects of reducing the sensitivity of the assay, reducing the binding or reducing the binding, and improving stability

Inactive Publication Date: 2007-03-08
ROCHE DIAGNOSTICS OPERATIONS INC
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0010] The C-terminal FKBP25 peptide FKBP25C, P109-D224, (J. Liang et al.) is also useful for a rapamycin assay. J. Liang et al. reported that “The N-terminal domain does not influence the binding specificity since the C-terminal domain shows the same binding preferences as the full length protein”. The N-terminal domain of FKBP25 (a˜13 kDa peptide proximal to P109) may lie near the C-40 position of bound rapamycin and may hinder or reduce the binding of the aminodextran rapamycin conjugated at the C-40 position and thus reduce the sensitivity of the assay. An advantage of using FKBP25C may be improved stability since the J. Liang authors state in the supplemental material for the paper, “Recombinant hFKBP25 is unstable and degrades rapidly following purification. With time, only a mixture of fragments with molecular weights from 13.7 to 15 kDa can be identified in the original FKBP25 sample.” Another potential advantage of FKBP25C may be a slight reduction in the pI as the N-terminal domain of FKBP25 is rich in basic amino acids (Galat et. al. 1992). A lower pI may reduce non-specific binding to proteins (with two pI's) in patient samples.
[0011] FKBP25 and FKBP25C are each useful in an assay for rapamycin as GST (glutathione S-transferase) fusion proteins. Fusion proteins containing the complete amino acid sequence of GST can form dimers (Amersham Biosciences, GST Gene Fusion System Handbook, page 27). GST is found in nature as a dimeric enzyme (D. B. Smith, K. S. Johnson, Gene, 67, 31, 1988). The advantage of using dimerized FKBP25 is to improve the sensitivity of an assay for rapamycin. The result of dimerization of FKBP25-GST or FKBP25C-GST fusion proteins is the formation of two binding sites per macromolecule, allowing cross-linking or binding to two identical ligands similar to an antibody. Dimerized FKBP25-GST or FKBP25C-GST fusion proteins can be used in solution to cross-link or agglutinate rapamycin coupled microparticles. Rapamycin in a sample can bind to the dimerized FKBP25-GST or FKBP25C-GST in solution and thus inhibit the agglutination of rapamycin coupled microparticles.

Problems solved by technology

For the drugs rapamycin and tacrolimus, a major challenge to the single receptor format is specificity since the class of immunophilin which binds to these drugs, the so-called FK binding proteins or FKBP's, generally do not distinguish to any significant degree between rapamycin and tacrolimus due to the structural similarities of the drugs.

Method used

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  • Single receptor assays for immunosuppressive drugs
  • Single receptor assays for immunosuppressive drugs

Examples

Experimental program
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specific embodiments

EXAMPLE 1

Preparation of rapamycin 40-O-p-nitrophenyl carbonate (1)

[0064] To 100 mg (0.109 mmol) of rapamycin was added 2.5 mL of freshly distilled dichloromethane (distilled over CaH2), 33 μL (0.40 mmol) of anhydrous pyridine and 33 mg (0.16 mmol) of p-nitrophenylchloroformate at −78° C. The reaction mixture was allowed to stir at −78° C. for 1 h and at room temperature for 1 h. The reaction was quenched with 50 mL of water at room temperature, and the resulting mixture was extracted with 4×50 mL of dichloromethane. The organic part was dried (anhydrous Na2SO4) and concentrated. The residue was purified by preparative HPLC using a gradient run of acetonitrile and water containing 0.1% trifluoroacetic acid. The fractions containing product were combined, concentrated in the rotary evaporator, and then lyophilized to give 82 mg (0.074 mmol, 69%) of rapamycin p-nitrophenyl carbonate (1) as a white solid. LC-MS: M+H 1101.5.

example 2

Preparation of 40-O-urethane Linked Rapamycin-Aminodextran Conjugate (2)

[0065] To 50 mg (1.25×10−3 mmol) of aminodextran (40,000) (prepared as in U.S. Pat. No. 6,653,456) was added 2 mL of anhydrous DMSO, and the mixture was allowed to stir for 5 minutes at room temperature. To the reaction mixture 10.7 mg (0.087 mmol) of 4-dimethylaminopyridine was added followed by a solution of 20 mg (0.018 mmol) of rapamycin p-nitrophenyl carbonate in 0.5 mL of anhydrous DMF. The reaction was allowed to stir at room temperature for 2 days. The resulting yellow solution was placed in a Spectrapor dialysis tubing (mw cut-off 2000) and dialyzed against 90% DMSO in DI water (2×3 h), 70% DMSO in DI water (1×3 hr), 50% DMSO in DI water (1×3 h), 30% DMSO in DI water (1×3 h), and 10% DMSO in DI water (1×3 h). This was followed by dialysis against DI water (6×6 h), and the resulting solution was lyophilized to give 39 mg of rapamycin-aminodextran conjugate (2) as a white solid. The incorporation of rapa...

example 3

Perparation of Rapamycin 40-O-glutarate NHS Ester (3)

[0066] A solution of 150 mg (0.164 mmol) of rapamycin in 4 mL of dichloromethane (freshly distilled over CaH2) was cooled to −78° C. To the reaction mixture was added 61 mg (0.25 mmol) of succinimido-oxycarbonyl-butyryl chloride, 15 mg (0.12 mmol) of 4-dimethylaminopyridine and 50 μL of pyridine. Succinimido-oxycarbonyl-butyryl chloride, i.e., 5-(2,5-dioxo-1-pyrrolidinyl-oxy)-5-oxo-pentanoyl chloride, was prepared according to Antonian et al., EP 0 503 454. The reaction mixture was allowed to stir at −78° C. for 2 h and LC-MS of the crude reaction mixture indicated incomplete reaction. To the reaction mixture an additional 61 mg (0.25 mmol) of succinimido oxycarbonyl butyryl chloride and 50 μL (0.60 mmol) of pyridine were added at −78° C. The resulting reaction mixture was allowed to stir at −78° C. for 2 h and then quenched with 50 mL of water. The aqueous layer was extracted with 5×50 mL of dichloromethane, and the organic laye...

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Abstract

A highly specific homogeneous assay method for an immunosuppressive drug using an immunophilin in a single receptor format is provided. In the simplest format, a single receptor is utilized analogous to a competitive immunoassay whereby an immunophilin is substituted for an antibody, and a competition results between a drug conjugate and the drug analyte for a limited number of immunophilin binding sites. In a microparticle agglutination assay format, an immunophilin is either bound to a particle or in solution. In the case where an immunophilin is bound to a particle, a polyvalent conjugate of the drug analyte is present in solution.

Description

FIELD OF THE INVENTION [0001] The present invention pertains to the field of therapeutic drug monitoring, and in particular, to assay for detecting and quantitating immunosuppressive drugs. BACKGROUND OF THE INVENTION [0002] Immunosuppressive drugs are used in immunosuppressive therapy to inhibit or prevent activity of the immune system. Clinically they are used to prevent the rejection of transplanted organs and tissues (e.g. bone marrow, heart, kidney, liver) and in the treatment of autoimmune diseases or diseases that are most likely of autoimmune origin (e.g. rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease, and ulcerative colitis). [0003] Single receptor assays for immunosuppressive drugs such as FK506 (tacrolimus) and rapamycin (sirolimus) have used immunophilins, e.g., FKBR's (FK binding proteins), but the FKBP's show significant cross-reactivity for more than one immunosuppressive drug or for inactive metabolites. Since immunosuppressive...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53
CPCG01N21/6445G01N21/76G01N2021/6432G01N33/54346G01N33/9493G01N33/5308
Inventor SIGLER, GERALDGHOSHAL, MITALIDORN, ALLANRASHID, SHAKER
Owner ROCHE DIAGNOSTICS OPERATIONS INC
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